WO2013141291A1 - Dispositif pour l'analyse cible de la streptavidine, et procédé d'analyse - Google Patents

Dispositif pour l'analyse cible de la streptavidine, et procédé d'analyse Download PDF

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WO2013141291A1
WO2013141291A1 PCT/JP2013/058042 JP2013058042W WO2013141291A1 WO 2013141291 A1 WO2013141291 A1 WO 2013141291A1 JP 2013058042 W JP2013058042 W JP 2013058042W WO 2013141291 A1 WO2013141291 A1 WO 2013141291A1
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nucleic acid
forming sequence
acid molecule
stem
catalytic
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PCT/JP2013/058042
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English (en)
Japanese (ja)
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克紀 堀井
金子　直人
穣 秋冨
信太郎 加藤
巌 和賀
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Ｎｅｃソフト株式会社
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Priority to US14/387,431 priority Critical patent/US9880161B2/en
Priority to EP13763690.8A priority patent/EP2829604A4/fr
Priority to JP2014506266A priority patent/JP6183917B2/ja
Publication of WO2013141291A1 publication Critical patent/WO2013141291A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/36—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Actinomyces; from Streptomyces (G)

Definitions

  • the present invention relates to a nucleic acid sensor for analysis of streptavidin, an analysis device, and an analysis method.
  • Streptavidin (hereinafter referred to as “SA”) is used as a research and testing substance in various fields such as clinical medicine, food, and environment due to its properties such as strong binding ability to biotin and high stability. Has been.
  • the target detection method the target is indirectly detected and quantified by detecting SA using the binding between the target, biotin and SA.
  • Non-Patent Document 1 Non-Patent Document 1
  • an object of the present invention is to provide a new sensor for detecting SA.
  • the nucleic acid sensor for analysis of the present invention is a nucleic acid sensor for analysis of SA, and has the following (I) and (I) having a catalytic nucleic acid molecule (D) that causes a catalytic function and a binding nucleic acid molecule (A) that binds to SA. II), (II ′) or (III) nucleic acid element.
  • first strand (ss1) the binding nucleic acid molecule (A), the loop forming sequence (L1) and the catalytic nucleic acid molecule (D) are linked in this order
  • second strand (ss2) a stem forming sequence (S A ), a loop forming sequence (L2) and a stem forming sequence (S D ) are linked in this order
  • the terminal region on the loop forming sequence (L1) side of the binding nucleic acid molecule (A) in the first strand (ss1) and the stem forming sequence (S A ) in the second strand (ss2) are complementary.
  • the terminal region on the loop-forming sequence (L1) side of the catalytic nucleic acid molecule (D) in the first strand (ss1) and the stem-forming sequence (S D ) in the second strand (ss2) are complementary.
  • the loop forming sequence (L1) in the first strand (ss1) and the loop forming sequence (L2) in the second strand (ss2) are non-complementary.
  • the catalytic function of the catalytic nucleic acid molecule (D) is inhibited by the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ),
  • binding of the SA and the binding nucleic acid molecule (A) releases the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ), and the catalytic nucleic acid.
  • the terminal region on the loop forming sequence (L1) side of the binding nucleic acid molecule (A) and the stem forming sequence (S A ) are complementary,
  • the terminal region on the loop forming sequence (L2) side of the catalytic nucleic acid molecule (D) and the stem forming sequence (S D ) are complementary,
  • the loop forming sequence (L1) and the loop forming sequence (L2) are non-complementary,
  • the catalytic function of the catalytic nucleic acid molecule (D) is inhibited by the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ),
  • binding of the SA and the binding nucleic acid molecule (A) releases the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ), and the catalytic nucleic acid.
  • the catalytic nucleic acid molecule (D), the loop forming sequence (L2), the stem forming sequence (S A ), the binding nucleic acid molecule (A), the loop forming sequence (L1) and the stem forming sequence (S D ) are in this order.
  • the terminal region on the loop forming sequence (L2) side of the catalytic nucleic acid molecule (D) and the stem forming sequence (S D ) are complementary,
  • the terminal region on the loop forming sequence (L1) side of the binding nucleic acid molecule (A) and the stem forming sequence (S A ) are complementary,
  • the loop forming sequence (L1) and the loop forming sequence (L2) are non-complementary,
  • the catalytic function of the catalytic nucleic acid molecule (D) is inhibited by the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ),
  • binding of the SA and the binding nucleic acid molecule (A) releases the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ), and the catalytic nucleic acid.
  • the analysis device of the present invention is an SA analysis device, and includes a base material, a nucleic acid sensor, and a detection unit.
  • the nucleic acid sensor and the detection unit are disposed on the base material, and the nucleic acid sensor is the book.
  • the detection unit is a detection unit that detects a catalytic function of the catalytic nucleic acid molecule (D) in the nucleic acid sensor.
  • the analysis method of the present invention is an analysis method of SA, the step of bringing a sample containing SA into contact with the nucleic acid sensor for SA analysis of the present invention, and the catalyst of the catalytic nucleic acid molecule (D) in the nucleic acid sensor It includes a step of detecting SA in the sample by detecting a function.
  • the analysis method of the present invention is an analysis method of SA, the step of bringing a sample containing SA into contact with the analysis device of the present invention, and the catalyst in the nucleic acid sensor in the detection unit of the analysis device It includes a step of detecting SA in the sample by detecting the catalytic function of the nucleic acid molecule (D).
  • the nucleic acid sensor of the present invention ON / OFF of the catalytic function of the catalytic nucleic acid molecule (D) can be switched depending on whether or not the binding nucleic acid molecule (A) and SA are bound. For this reason, the presence or amount of SA can be easily detected by detecting the catalytic function of the catalytic nucleic acid molecule (D).
  • the analysis device of the present invention uses the nucleic acid sensor as described above, for example, the device can be reduced in size and chipped, and a simple analysis can be performed even for a large number of samples. . For this reason, the present invention can be said to be an extremely useful technique for research and examination in various fields such as clinical medicine, food, and environment.
  • “analysis” is a concept including, for example, quantitative analysis, semi-quantitative analysis, and qualitative analysis.
  • FIG. 1 is a schematic diagram showing an example of a nucleic acid element in the nucleic acid sensor of the present invention.
  • FIG. 2 is a schematic diagram showing another example of the nucleic acid element in the nucleic acid sensor of the present invention.
  • FIG. 3 is a schematic diagram showing another example of the nucleic acid element in the nucleic acid sensor of the present invention.
  • FIG. 4 is a schematic diagram showing another example of the nucleic acid element in the nucleic acid sensor of the present invention.
  • FIG. 5 (A) is a photograph of the reaction solution in Example 1
  • FIG. 5 (B) is a graph showing the results of absorbance measurement in Example 1 described above.
  • FIG. 6 is a graph showing the results of absorbance measurement in Example 2.
  • FIG. 7 is a graph showing the results of absorbance measurement in Example 3.
  • the nucleic acid sensor for analysis of SA of the present invention comprises the above-mentioned (I) and (II) having the catalytic nucleic acid molecule (D) that causes a catalytic function and the binding nucleic acid molecule (A) that binds to SA. (II ') or (III) nucleic acid element.
  • binding nucleic acid molecule (A) is not particularly limited as long as it is a nucleic acid molecule that binds to SA.
  • binding to SA may be capable of binding to any of SA fragments, SA derivatives, and SA derivatives, for example.
  • the binding nucleic acid molecule (A) is, for example, a single strand.
  • the length of the binding nucleic acid molecule (A) is not particularly limited, and the lower limit is, for example, 18 base length, preferably 20 base length, more preferably 24 base length, and the upper limit is, for example, The length is 120 bases, preferably 85 bases, more preferably 60 bases, and even more preferably 26 bases.
  • binding nucleic acid molecule (A) examples include those containing the following polynucleotide (a1), (a2), (a3) or (a4).
  • the binding nucleic acid molecule (A) may be, for example, a molecule composed of the polynucleotide or a molecule containing the polynucleotide.
  • the binding nucleic acid molecule (A) containing the polynucleotide (a1), (a2), (a3) or (a4) can also be referred to as a binding DNA molecule, for example.
  • A1 a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 10 (a2) in the nucleotide sequence of (a1), one or more bases are substituted, deleted, added and / or inserted A polynucleotide comprising a base sequence and binding to SA (a3) A polynucleotide comprising a base sequence having 50% or more identity to the base sequence of (a1) and capable of binding to SA (a4) A polynucleotide comprising a base sequence complementary to the base sequence hybridizing under stringent conditions with the base sequence of (a1) and capable of binding to SA
  • “one or more” is not particularly limited, and the polynucleotide (a2) may be bound to SA.
  • the number of the substituted bases is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, still more preferably 1 or 2. Particularly preferred is one.
  • the number of added or inserted bases is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and still more preferably 1 or 2.
  • the number of deleted bases is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and further preferably 2 or 1. Particularly preferred is one.
  • the identity is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, 96% or more with respect to the base sequence (a1). 97% or more, 98% or more, particularly preferably 99% or more.
  • the identity can be calculated, for example, by calculating under default conditions using BLAST or the like.
  • hybridizes under stringent conditions is, for example, well-known experimental conditions for hybridization by those skilled in the art.
  • the “stringent conditions” are, for example, that hybridization is performed at 60 to 68 ° C. in the presence of 0.7 to 1 mol / L NaCl, and then 0.1 to 2 times the SSC solution is used.
  • Conditions under which the nucleotide sequence can be identified by washing at 65 to 68 ° C. 1 ⁇ SSC consists of 150 mmol / L NaCl, 15 mmol / L sodium citrate.
  • the binding nucleic acid molecule (A) is not limited to these examples, and may be any nucleic acid molecule that binds to SA as described above.
  • the binding nucleic acid molecule (A) is, for example, a molecule containing a nucleotide residue, and may be a molecule consisting only of a nucleotide residue or a molecule containing a nucleotide residue.
  • the nucleotide is, for example, ribonucleotide, deoxyribonucleotide and derivatives thereof.
  • the binding nucleic acid molecule (A) may contain, for example, only one kind of ribonucleotide, deoxyribonucleotide and derivatives thereof, two kinds or more, or all of them.
  • the nucleic acid molecule may be, for example, DNA containing deoxyribonucleotide and / or a derivative thereof, RNA containing ribonucleotide and / or a derivative thereof, or a chimera (DNA / RNA containing the former and the latter )
  • the nucleotide may contain, for example, either a natural base (non-artificial base) or a non-natural base (artificial base) as a base.
  • a natural base include A, C, G, T, U, and modified bases thereof.
  • the modification include methylation, fluorination, amination, and thiolation.
  • the unnatural base include 2′-fluoropyrimidine, 2′-O-methylpyrimidine and the like. Specific examples include 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, Examples include 2-thiouracil.
  • the nucleotide may be, for example, a modified nucleotide, and the modified nucleotide is, for example, a 2′-methylated-uracil nucleotide residue, 2′-methylated-cytosine nucleotide residue, 2′-fluorinated-uracil nucleotide. Residue, 2′-fluorinated-cytosine nucleotide residue, 2′-aminated-uracil nucleotide residue, 2′-aminated-cytosine nucleotide residue, 2′-thiolated-uracil nucleotide residue, 2′- Thio-cytosine nucleotide residues and the like.
  • the binding nucleic acid molecule (A) may include non-nucleotides such as PNA (peptide nucleic acid) and LNA (Locked Nucleic Acid).
  • the catalytic nucleic acid molecule (D) may be a nucleic acid molecule that causes a catalytic function.
  • the catalytic function is, for example, a catalytic function of a redox reaction.
  • the oxidation-reduction reaction may be a reaction that causes transfer of electrons between two substrates in the process of generating a product from the substrates, for example.
  • the kind of the redox reaction is not particularly limited.
  • the catalytic function of the oxidation-reduction reaction includes, for example, the same activity as an enzyme, and specifically includes, for example, the same activity as peroxidase (hereinafter referred to as “peroxidase-like activity”).
  • the peroxidase activity examples include horseradish peroxidase (HRP) activity.
  • the catalytic nucleic acid molecule (D) can be referred to as a DNA enzyme or DNAzyme in the case of DNA as described later, and can be referred to as an RNA enzyme or RNAzyme in the case of RNA as described later.
  • the catalytic nucleic acid molecule (D) is preferably a nucleic acid forming a G-quartet (or G-tetrad) structure, more preferably a nucleic acid forming a guanine quadruplex (or G-quadruplex) structure.
  • the G-tetrad is, for example, a surface structure in which guanine is a tetramer
  • the G-quadruplex is, for example, a structure in which a plurality of the G-tetrads are overlapped.
  • the G-tetrad and the G-quadruplex are formed, for example, in a nucleic acid that repeatedly has a G-rich structural motif.
  • the G-tetrad examples include a parallel type and an anti-parallel type, and a parallel type is preferable.
  • the catalytic nucleic acid molecule (D) inhibits the formation of the G-tetrad by forming the stem in a state where SA is not bound to the binding nucleic acid element (A).
  • SA is bound to the binding nucleic acid molecule (A) to release the stem formation and form the G-tetrad.
  • the catalytic nucleic acid molecule (D) is preferably a nucleic acid that can bind to porphyrin, and specifically, a nucleic acid that forms the G-tetrad and can bind to the porphyrin. It is known that the nucleic acid having G-tetrad generates a catalytic function of the oxidation-reduction reaction as described above, for example, by binding to the porphyrin to form a complex. In the nucleic acid element, the catalytic nucleic acid molecule (D) is inhibited from binding to the porphyrin, for example, by forming the stem in a state where SA is not bound to the binding nucleic acid molecule (A).
  • the binding of SA to the binding nucleic acid molecule (A) releases the stem formation and binds to the porphyrin.
  • the catalytic nucleic acid molecule (D) inhibits the formation of the G-tetrad in a state where, for example, SA is not bound to the binding nucleic acid molecule (A), and It is preferable that binding to the porphyrin is inhibited and SA binds to the binding nucleic acid molecule (A) to form the G-tetrad and bind to the porphyrin.
  • the porphyrin is not particularly limited, and examples thereof include unsubstituted porphyrin and derivatives thereof.
  • examples of the derivatives include substituted porphyrins and metal porphyrins complexed with metal elements.
  • Examples of the substituted porphyrin include N-methylmesoporphyrin.
  • Examples of the metal porphyrin include hemin, which is a trivalent iron complex.
  • the porphyrin is, for example, preferably the metal porphyrin, more preferably hemin.
  • the catalytic nucleic acid molecule (D) is, for example, a single strand.
  • the length of the catalyst nucleic acid molecule (D) is not particularly limited, and the lower limit is, for example, 11 bases, preferably 13 bases, more preferably 15 bases, and the upper limit is, for example, It is 60 bases long, preferably 36 bases long, more preferably 18 bases long.
  • Examples of the catalytic nucleic acid molecule (D) include DNAzymes disclosed in the following papers (1) to (4) as DNA having peroxidase activity.
  • Tao et al. Anal. Chem., 2009, vol.81, p.2144-2149
  • catalytic nucleic acid molecule (D) examples include those containing the following polynucleotide (d1), (d2), (d3) or (d4).
  • the catalytic nucleic acid molecule (D) may be, for example, a molecule composed of the polynucleotide or a molecule containing the polynucleotide.
  • the catalytic nucleic acid molecule (D) containing the polynucleotide (d1), (d2), (d3) or (d4) can also be referred to as, for example, a catalytic DNA molecule or DNAzyme.
  • (D1) a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 11 to 31 and 61 to 80 (d2) in the nucleotide sequence of (d1), one or more bases are substituted, deleted, added and / or Or consisting of an inserted base sequence, and a polynucleotide (d3) that causes a catalytic function of the oxidation-reduction reaction (d3) consisting of a base sequence having 50% or more identity with the base sequence, and The polynucleotide (d4) that causes the catalytic function of the redox reaction, the base sequence complementary to the base sequence that hybridizes with the base sequence of the (d1) under stringent conditions, and the redox reaction Polynucleotides that cause the catalytic function of
  • the number of the substituted bases is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, further preferably 1 or 2. Particularly preferred is one.
  • the number of added or inserted bases is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, further preferably 1 or 2 in the base sequence (d1).
  • the number of deleted bases is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and further preferably 2 or 1. Particularly preferred is one.
  • the identity is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, 96% or more with respect to the base sequence (d1). 97% or more, 98% or more, particularly preferably 99% or more.
  • the identity can be calculated, for example, by calculating under default conditions using BLAST or the like.
  • the catalytic nucleic acid molecule (D) is not limited to the examples (d1) to (d4) described above, and may be any nucleic acid molecule that causes the catalytic function as described above.
  • the catalyst nucleic acid molecule (D) is, for example, a molecule containing a nucleotide residue, and may be a molecule consisting only of a nucleotide residue or a molecule containing a nucleotide residue.
  • the nucleotide is the same as described above.
  • the catalytic nucleic acid molecule (D) may contain, for example, only one kind of ribonucleotide, deoxyribonucleotide and derivatives thereof, two kinds or more, or all of them.
  • the nucleic acid molecule may be, for example, DNA containing deoxyribonucleotide and / or a derivative thereof, RNA containing ribonucleotide and / or a derivative thereof, or a chimera (DNA / RNA containing the former and the latter )
  • the nucleotide can be exemplified by the example in the binding nucleic acid molecule (A).
  • the catalytic nucleic acid molecule (D) may contain non-nucleotides such as PNA (peptide nucleic acid) and LNA (Locked Nucleic Acid).
  • examples of the nucleic acid element include (I), (II), (II ′) and (III) as described above. These three forms will be described below, but each form can incorporate the description unless otherwise indicated.
  • the nucleic acid element (I) is (I) a double-stranded nucleic acid element composed of a first strand and a second strand.
  • the second chain is also called a block chain.
  • the first strand (ss1) the binding nucleic acid molecule (A), the loop-forming sequence (L1) and the catalytic nucleic acid molecule (D) are linked in this order
  • the second strand (ss2) is The stem forming sequence (S A ), the loop forming sequence (L2) and the stem forming sequence (S D ) are linked in this order.
  • the terminal region on the loop forming sequence (L1) side of the binding nucleic acid molecule (A) in the first strand (ss1) and the stem forming sequence (S A ) in the second strand (ss2) are complementary.
  • the terminal region on the loop forming sequence (L1) side of the catalytic nucleic acid molecule (D) in the first strand (ss1) and the stem forming sequence (S D ) in the second strand (ss2) are complementary.
  • the loop forming sequence (L1) in the first strand (ss1) and the loop forming sequence (L2) in the second strand (ss2) are non-complementary.
  • the catalytic function of the catalytic nucleic acid molecule (D) is inhibited by the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ).
  • the inhibition of the catalytic function occurs, for example, when the catalytic nucleic acid molecule is caged by this stem formation. That is, due to the formation of the stem, the catalytic nucleic acid molecule (D) cannot take a structure that causes a catalytic function, thereby inhibiting the catalytic function.
  • the catalytic function of the catalytic nucleic acid molecule (D) occurs.
  • the occurrence of the catalytic function occurs, for example, when stem formation is released and cage formation of the catalytic nucleic acid molecule is released.
  • the catalytic function is generated by releasing the formation of the stem so that the catalytic nucleic acid molecule (D) has an original structure that generates the catalytic function. Note that the present invention is not limited to these mechanisms.
  • the catalytic function of the catalytic nucleic acid molecule (D) is inhibited by the stem formation (switch OFF), and when SA is present, the stem The formation is released and the catalytic function of the catalytic nucleic acid molecule (D) occurs (switch ON).
  • the nucleic acid element (I) for example, in the absence of SA, the terminal region on the loop forming sequence (L1) side of the binding nucleic acid molecule (A) in the first strand (ss1), The stem-forming sequence (S A ) in the second strand (ss2) forms a stem, and the terminal region on the loop-forming sequence (L1) side of the catalytic nucleic acid molecule (D) in the first strand (ss1)
  • the stem forming sequence (S D ) in the second strand (ss2) forms a stem, and the loop forming sequence (L1) and the loop forming sequence (L2) are between the two stems.
  • “complementary” means, for example, that two regions to be aligned may be completely complementary, or may be complementary to the extent that a stem can be formed (hereinafter the same).
  • “non-complementary” means, for example, that the two regions to be aligned may be completely non-complementary or non-complementary to the extent that an internal loop can be formed. (The same applies hereinafter).
  • FIG. 1 shows the state of the nucleic acid element (I) in the absence of SA.
  • 1A and 1B show a form in which the directions of the first chain (ss1) and the second chain (ss2) are opposite to each other.
  • an arrow indicates a direction from the 5 'side to the 3' side (hereinafter the same).
  • the upper strand is the first strand (ss1)
  • A is the binding nucleic acid molecule (A)
  • L1 is the loop-forming sequence (L1)
  • D is the catalytic nucleic acid molecule (D )
  • the lower strand is the second strand (ss2)
  • S A is the stem-forming sequence (S A )
  • L2 is the loop-forming sequence (L2)
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • S D is the stem
  • a stem is formed between the terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (S A ), and the catalytic nucleic acid molecule (D)
  • a stem is formed between the terminal region on the loop-forming sequence (L1) side and the stem-forming sequence (S D ), and between the loop-forming sequence (L1) and the loop-forming sequence (L2).
  • An internal loop is formed.
  • the direction of each component is not particularly limited.
  • the first strand (ss1) includes, for example, the binding nucleic acid molecule (A), the loop-forming sequence (L1), and the catalytic nucleic acid molecule (D) from the 5 ′ side.
  • the second strand (ss2) is linked in this order, and the stem-forming sequence (S A ), the loop-forming sequence (L2) and the stem-forming sequence (S D ) are arranged in this order from the 3 ′ side. It is preferable to connect with.
  • the 3 ′ terminal region of the binding nucleic acid molecule (A) in the first strand (ss1) and the stem-forming sequence (S A ) in the second strand (ss2) are complementary
  • the 5 ′ terminal region of the catalytic nucleic acid molecule (D) in the first strand (ss1) and the stem-forming sequence (S D ) in the second strand (ss2) are preferably complementary. As shown in FIG.
  • the first strand (ss1) is, for example, from the 3 ′ side, the binding nucleic acid molecule (A), the loop-forming sequence (L1) and the catalytic nucleic acid molecule (D ) Are linked in this order, and the second strand (ss2), from the 5 ′ side, the stem-forming sequence (S A ), the loop-forming sequence (L2) and the stem-forming sequence (S D ) You may connect in this order.
  • the 5 ′ end region of the binding nucleic acid molecule (A) in the first strand (ss1) and the stem-forming sequence (S A ) in the second strand (ss2) are complementary
  • the 3 ′ terminal region of the catalytic nucleic acid molecule (D) in the first strand (ss1) and the stem-forming sequence (S D ) in the second strand (ss2) are complementary.
  • the nucleic acid element (I) is presumed to be able to detect excellent sensitivity by forming an internal loop in this way, but the present invention is not limited to this assumption.
  • the length of the inner loop is not particularly limited.
  • the loop forming sequence (L1) in the first strand (ss1) and the loop forming sequence (L2) in the second strand (ss2) are, for example, 0 to 30 bases in length, preferably 1 to The length is 30 bases, more preferably 1 to 15 bases, and even more preferably 1 to 6 bases.
  • the lengths of the loop forming sequence (L1) and the loop forming sequence (L2) may be the same or different, for example. In the latter case, the difference in length is not particularly limited, and is, for example, 1 to 10 bases long, preferably 1 or 2 bases long, more preferably 1 base long.
  • the nucleic acid element (I) may have only one of the loop forming sequence (L1) and the loop forming sequence (L2).
  • the nucleic acid element (I) is presumed to be able to detect excellent sensitivity by forming an internal loop in this way, but the present invention is not limited to this assumption.
  • the length of each stem is not particularly limited.
  • the length of the stem can be adjusted by, for example, the length of the stem-forming sequence (S A ) and the stem-forming sequence (S D ) in the second strand (ss2).
  • the length of the stem-forming sequence (S A ) is, for example, 0 to 60 bases long, 1 to 60 bases long, preferably 0 to 10 bases long, 1 to 10 bases long.
  • the length of the stem-forming sequence (S D ) is, for example, 0-30 bases long, 1-30 bases long, more preferably 0-10 bases long, 1-10 bases long, More preferably, the length is 1 to 6 bases.
  • the stem forming sequence (S A ) and the stem forming sequence (S D ) may have the same length, the former may be long, or the latter may be long.
  • the lengths of the first strand (ss1) and the second strand (ss2) are not particularly limited.
  • the length of the first strand (ss1) is, for example, 40 to 200 bases, preferably 42 to 100 bases, and more preferably 45 to 60 bases.
  • the length of the second strand (ss2) is, for example, 4 to 120 bases, preferably 5 to 25 bases, more preferably 10 to 15 bases.
  • the nucleic acid element (I) is shown below, but the present invention is not limited thereto.
  • the first strand (ss1) is exemplified below.
  • the underlined portion on the 5 ′ side is the SA aptamer of SEQ ID NO: 5 (A in FIG. 1A), and the poly dT is the loop-forming sequence (L1 in FIG. 1A).
  • the underlined portion is DNAzyme of SEQ ID NO: 18 (neco0584) (D in FIG. 1A).
  • SA.neco.D3A2 SEQ ID NO: 32) 5'- CCGACGCACCGATCGCAGGTTCGG TTTTTTTTTT GGGTGGGAGGGTCGGG -3 '
  • the second strand (ss2) with respect to the first strand (ss1) is exemplified below.
  • the underlined portion on the 5 ′ side is the stem-forming sequence ( SD in FIG. 1A) complementary to the 5 ′ side region of the DNAzyme of the first strand (ss1)
  • the poly dT is The loop-forming sequence (L2 in FIG. 1 (A))
  • the underlined portion on the 3 ′ side is complementary to the 3′-side region of the SA aptamer of the first strand (ss1) (FIG. 1 ( In A), S A ).
  • the nucleic acid element (II) is a single-stranded nucleic acid element, and the binding nucleic acid molecule (A), the loop forming sequence (L1), the stem forming sequence (S D ), the catalyst The nucleic acid molecule (D), the loop forming sequence (L2) and the stem forming sequence (S A ) are linked in this order.
  • the terminal region on the loop forming sequence (L1) side of the binding nucleic acid molecule (A) and the stem forming sequence (S A ) are complementary, and the loop forming sequence (L2) of the catalytic nucleic acid molecule (D)
  • the terminal region on the side and the stem forming sequence (S D ) are complementary, and the loop forming sequence (L1) and the loop forming sequence (L2) are non-complementary.
  • the nucleic acid element (II) is a catalyst of the catalytic nucleic acid molecule (D) by the respective stem formation in the stem forming sequence (S A ) and the stem forming sequence (S D ) in the absence of SA. Function is inhibited.
  • the inhibition of the catalytic function occurs, for example, when the catalytic nucleic acid molecule is caged by such self-association stem formation. That is, due to the formation of the stem, the catalytic nucleic acid molecule (D) cannot take the original structure that causes the catalytic function, thereby inhibiting the catalytic function.
  • the catalytic function of the catalytic nucleic acid molecule (D) occurs.
  • the occurrence of the catalytic function occurs, for example, when the stem formation due to self-association is released and the catalytic nucleic acid molecule is released from the cage.
  • the catalytic function is generated by releasing the formation of the stem so that the catalytic nucleic acid molecule (D) has an original structure that generates the catalytic function. Note that the present invention is not limited to these mechanisms.
  • nucleic acid element (II) As in the case of the nucleic acid element (I), when SA is absent, the catalytic function of the catalytic nucleic acid molecule (D) is inhibited by the stem formation (switch OFF), and the SA When present, the stem formation is released, and the catalytic function of the catalytic nucleic acid molecule (D) is generated (switch ON).
  • the nucleic acid element (II) includes, for example, a terminal region on the loop forming sequence (L1) side of the binding nucleic acid molecule (A) in the absence of SA, and the stem forming sequence (S A ) Form a stem, and the terminal region on the loop-forming sequence (L2) side of the catalytic nucleic acid molecule (D) and the stem-forming sequence (S D ) form a stem, and the loop-forming sequence (L1) And the loop forming sequence (L2) form an internal loop between the two stems.
  • the state of the nucleic acid element (II) in the absence of the SA is shown in the schematic diagram of FIG. 2A and 2B show a form in which the directions are opposite to each other.
  • A is the binding nucleic acid molecule (A)
  • L1 is the loop-forming sequence (L1)
  • SD is the stem-forming sequence (S D )
  • D is the catalytic nucleic acid molecule (D)
  • L2 represents the loop forming sequence (L2)
  • S A represents the stem forming sequence (S A ).
  • stems are formed at two locations by self-annealing of the nucleic acid element (II), and an internal loop is formed between the stems.
  • a stem is formed between the terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (S A ), and the catalytic nucleic acid molecule (D)
  • a stem is formed between the terminal region on the loop-forming sequence (L2) side and the stem-forming sequence (S D ), and between the loop-forming sequence (L1) and the loop-forming sequence (L2).
  • An internal loop is formed.
  • the direction of each component is not particularly limited. As shown in FIG. 2 (B), the nucleic acid element (II) is, for example, from the 3 ′ side, the binding nucleic acid molecule (A), the loop-forming sequence (L1), the stem-forming sequence (S D ), The catalytic nucleic acid molecule (D), the loop forming sequence (L2) and the stem forming sequence (S A ) are preferably linked in this order.
  • the 5 ′ end region of the binding nucleic acid molecule (A) and the stem-forming sequence (S A ) are complementary, and the 5 ′ end region of the catalytic nucleic acid molecule (D) and the stem formation
  • the sequence (S D ) is preferably complementary.
  • the nucleic acid element (II) has, for example, the binding nucleic acid molecule (A), the loop-forming sequence (L1), and the stem-forming sequence ( SD ) from the 5 ′ side. ),
  • the catalytic nucleic acid molecule (D), the loop forming sequence (L2) and the stem forming sequence (S A ) may be linked in this order.
  • the 3 ′ end region of the binding nucleic acid molecule (A) and the stem-forming sequence (S A ) are complementary, and the 3 ′ end region of the catalytic nucleic acid molecule (D) and the stem formation
  • the sequence (S D ) is preferably complementary.
  • the length of the inner loop is not particularly limited.
  • the loop-forming sequence (L1) and the loop-forming sequence (L2) are each, for example, 0 to 30 bases long, preferably 1 to 30 bases long, more preferably 1 to 15 bases long More preferably, the length is 1 to 6 bases.
  • the lengths of the loop forming sequence (L1) and the loop forming sequence (L2) may be the same or different, for example. In the latter case, the difference in length is not particularly limited, and is, for example, 1 to 10 bases long, preferably 1 or 2 bases long, more preferably 1 base long.
  • the nucleic acid element (II) may have only one of the loop forming sequence (L1) and the loop forming sequence (L2). The nucleic acid element (II) is presumed to be able to detect excellent sensitivity by forming an internal loop in this way, but the present invention is not limited to this assumption.
  • the length of each stem is not particularly limited.
  • the length of the stem can be adjusted by, for example, the length of the stem forming sequence (S A ) and the stem forming sequence (S D ).
  • the length of the stem forming sequence (S A ) is, for example, 0 to 60 bases long, 1 to 60 bases long, preferably 1 to 10 bases long, more preferably 1 to 7 bases long.
  • the length of the stem-forming sequence (S D ) is, for example, 0 to 30 bases long, 1 to 30 bases long, preferably 0 to 10 bases long, 1 to 10 bases long, more preferably 0 to 7 bases long and 1-7 bases long.
  • the stem forming sequence (S A ) and the stem forming sequence (S D ) may have the same length, the former may be long, or the latter may be long.
  • the length of the nucleic acid element (II) is not particularly limited.
  • the length of the nucleic acid element (II) is, for example, 40 to 120 bases, preferably 45 to 100 bases, and more preferably 50 to 80 bases.
  • the nucleic acid element (II) is shown below, but the present invention is not limited thereto.
  • the lower case sequence is the stem formation sequence (S A in FIG. 2B)
  • the upper case poly dT is the loop formation sequence (L2 in FIG. 2B)
  • the underlined portion is DNAzyme of SEQ ID NO: 18 (neco0584) (D in FIG. 2B)
  • the lower case sequence is the stem forming sequence (S D in FIG. 2B)
  • the upper case poly dT is the loop forming sequence (FIG. 2 (B) is L1)
  • the underlined portion is the SA aptamer of SEQ ID NO: 5 (A in FIG. 2 (B)).
  • the nucleic acid element (II ′) is different from the nucleic acid element (II) in the binding nucleic acid molecule (A), the catalytic nucleic acid molecule (D), the stem-forming sequence (S A ), and the stem formation.
  • the sequence (S D ), the loop-forming sequence (L1), and the loop-forming sequence (L2) are single-stranded nucleic acid elements in a positional relationship in which they are interchanged.
  • the nucleic acid element (II ′) can be referred to the description of the nucleic acid element (II) unless otherwise specified.
  • the nucleic acid element (II ′) includes the catalytic nucleic acid molecule (D), the loop forming sequence (L2), the stem forming sequence (S A ), the binding nucleic acid molecule (A), and the loop forming sequence (L1). ) And stem-forming sequence (S D ) are linked in this order.
  • the terminal region on the loop forming sequence (L2) side of the catalytic nucleic acid molecule (D) and the stem forming sequence (S D ) are complementary, and the loop forming sequence (L1) of the binding nucleic acid molecule (A)
  • the terminal region on the side and the stem-forming sequence (S A ) are complementary.
  • the nucleic acid element (II ′) can form the catalytic nucleic acid molecule (D) by forming each stem in the stem-forming sequence (S A ) and the stem-forming sequence (S D ) in the absence of SA.
  • the catalytic function is impaired.
  • the inhibition of the catalytic function occurs, for example, when the catalytic nucleic acid molecule is caged by such self-association stem formation. That is, due to the formation of the stem, the catalytic nucleic acid molecule (D) cannot take the original structure that causes the catalytic function, thereby inhibiting the catalytic function.
  • the catalytic function of the catalytic nucleic acid molecule (D) occurs.
  • the occurrence of the catalytic function occurs, for example, when the stem formation due to self-association is released and the catalytic nucleic acid molecule is released from the cage.
  • the catalytic function is generated by releasing the formation of the stem so that the catalytic nucleic acid molecule (D) has an original structure that generates the catalytic function. Note that the present invention is not limited to these mechanisms.
  • the nucleic acid element (II ′) when SA is absent, the catalytic function of the catalytic nucleic acid molecule (D) is inhibited by the stem formation (switch OFF), and SA Is present, the stem formation is released, and the catalytic function of the catalytic nucleic acid molecule (D) occurs (switch ON).
  • the nucleic acid element (II ′) includes, for example, a terminal region on the loop-forming sequence (L1) side of the binding nucleic acid molecule (A) in the absence of SA, and the stem-forming sequence (S A ).
  • the state of the nucleic acid element (II ′) in the absence of the SA is shown in the schematic diagram of FIG. 3A and 3B show a form in which the directions are opposite to each other.
  • A is the binding nucleic acid molecule (A)
  • L1 is the loop forming sequence (L1)
  • D is the catalytic nucleic acid molecule
  • L2 is the loop forming sequence (L2)
  • S A Indicates the stem-forming sequence (S A )
  • SD indicates the stem-forming sequence (S D ).
  • stems are formed at two locations by self-annealing of the nucleic acid element (II ′), and an internal loop is formed between the stems.
  • a stem is formed between the terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (S A ), and the catalytic nucleic acid molecule (D)
  • a stem is formed between the terminal region on the loop-forming sequence (L2) side and the stem-forming sequence (S D ), and between the loop-forming sequence (L1) and the loop-forming sequence (L2).
  • An internal loop is formed.
  • the direction of each component is not particularly limited. As shown in FIG. 3 (B), the nucleic acid element (II ′) is, for example, from the 3 ′ side, the catalytic nucleic acid molecule (D), the loop-forming sequence (L2), and the stem-forming sequence (S A ).
  • the binding nucleic acid molecule (A), the loop forming sequence (L1), and the stem forming sequence (S D ) are preferably linked in this order.
  • the 5 ′ end region of the binding nucleic acid molecule (A) and the stem-forming sequence (S A ) are complementary, and the 5 ′ end region of the catalytic nucleic acid molecule (D) and the stem formation
  • the sequence (S D ) is preferably complementary.
  • the nucleic acid element (II ′) has, for example, the catalytic nucleic acid molecule (D), the loop forming sequence (L2), and the stem forming sequence (S A ), the binding nucleic acid molecule (A), the loop forming sequence (L1), and the stem forming sequence (S D ) may be linked in this order.
  • the 3 ′ end region of the binding nucleic acid molecule (A) and the stem-forming sequence (S A ) are complementary, and the 3 ′ end region of the catalytic nucleic acid molecule (D) and the stem formation
  • the sequence (S D ) is preferably complementary.
  • the nucleic acid element (III) is a single-stranded nucleic acid element, and the catalytic nucleic acid molecule (D), the intervening sequence (I), and the binding nucleic acid molecule (A) are arranged in this order.
  • the intervening sequence (I) is non-complementary to the catalytic nucleic acid molecule (D) and the binding nucleic acid molecule (A).
  • SA the catalytic function of the catalytic nucleic acid molecule
  • the catalytic function of the catalytic nucleic acid molecule (D) occurs.
  • the nucleic acid element (III) like the nucleic acid elements (I), (II) and (II ′), inhibits the catalytic function of the catalytic nucleic acid molecule (D) when SA is absent (switch OFF). ), When SA is present, the catalytic function of the catalytic nucleic acid molecule (D) occurs (switch ON).
  • FIG. 4 shows the state of the nucleic acid element (III) in the absence of SA.
  • A indicates the binding nucleic acid molecule (A)
  • I indicates the intervening sequence (I)
  • D indicates the catalytic nucleic acid molecule (D).
  • 4A and 4B show a form in which the directions are opposite to each other.
  • the catalytic function of the catalytic nucleic acid molecule (D) is inhibited in the absence of SA, and the catalytic function of the catalytic nucleic acid molecule (D) is generated in the presence of SA.
  • the following reason is presumed. The present invention is not limited to these assumptions.
  • a part of the catalytic nucleic acid molecule (D) and a part of the binding nucleic acid molecule (A) interact, and the catalytic nucleic acid molecule (D) It is presumed that a non-G quartet structure is formed.
  • the main form of the nucleic acid element (III) is tilted to the original structure of the bound nucleic acid element (A), that is, the three-dimensional structure that binds to SA, resulting in an overall structural change. It is considered that the catalytic nucleic acid molecule (D) forms a G quartet structure, and its catalytic function occurs.
  • the direction of each component is not particularly limited.
  • the nucleic acid element (III) includes, for example, the catalytic nucleic acid molecule (D), the intervening sequence (I) and the binding nucleic acid molecule (A) from the 5 ′ side. It is preferable to connect in order.
  • the nucleic acid element (III) includes, for example, the catalytic nucleic acid molecule (D), the intervening sequence (I), and the binding nucleic acid molecule (A) from the 3 ′ side. These may be connected in this order.
  • the length of the intervening sequence (I) is not particularly limited.
  • the length of the intervening sequence (I) is, for example, 0 to 30 bases or 1 to 30 bases long, preferably 0 to 10 bases or 1 to 10 bases, more preferably 0 to 8 bases. Length is 1 to 8 bases.
  • the length of the nucleic acid element (III) is not particularly limited.
  • the length of the nucleic acid element (III) is, for example, 30 to 120 bases, preferably 35 to 80 bases, and more preferably 40 to 60 bases.
  • nucleic acid element (III) Specific examples of the nucleic acid element (III) are shown below, but the present invention is not limited thereto.
  • the underlined portion on the 5 ′ side is the DNAzyme of SEQ ID NO: 11 (EAD2) (D in FIG. 4 (A)), and the underlined portion on the 3 ′ side is that of SEQ ID NO: 5 It is an SA aptamer (A in FIG. 4), and the poly dT between them is an intervening sequence (I in FIG. 4 (A)).
  • SA.neco.D0.A0 the underlined portion on the 5 ′ side is the DNAzyme of SEQ ID NO: 18 (neco0584) (D in FIG.
  • each region may be, for example, a direct connection or an indirect connection.
  • each region is connected by a phosphodiester bond.
  • region connects through an intervening linker, for example.
  • the intervening linker include nucleic acid molecules composed of nucleotides and / or non-nucleotides as described above.
  • the intervening linker is preferably, for example, a single chain.
  • the nucleic acid sensor of the present invention may be, for example, a sensor composed only of the nucleic acid element or a sensor including other components.
  • the nucleic acid sensor of the present invention can also be referred to as a device for detecting SA, for example.
  • the other component include a base material on which the nucleic acid element is disposed.
  • the base material include substrates such as substrates, beads, and tubes.
  • Other examples of the other component include a linker.
  • the linker can be used, for example, for linking the nucleic acid element and the base material when the nucleic acid element is immobilized on the base material.
  • the analytical device of the present invention described later can be referred to.
  • connection site with the linker is not particularly limited, and for example, any end of the nucleic acid element is preferable.
  • the double-stranded nucleic acid element (I) includes, for example, either end of the first strand (ss1) having the binding nucleic acid molecule (A) and the catalytic nucleic acid molecule (D), and / or the first strand. Either end of the two strands (ss2) is preferred.
  • the single-stranded nucleic acid elements (II), (II ′) and (III) are preferably at either end, for example.
  • the linker is also referred to as a terminal linker. Examples of the linker include nucleic acid molecules composed of nucleotides and / or non-nucleotides as described above.
  • the terminal linker is preferably a single chain, for example.
  • the nucleic acid element may be used in a free state, or may be used in a state where the nucleic acid element is immobilized. In the latter case, for example, it can be immobilized on the substrate and used as a device.
  • the method for using the nucleic acid sensor of the present invention is not particularly limited, and can be used for the SA analysis method of the present invention as follows.
  • the analysis method of the present invention is an analysis method of SA, the contacting step of bringing the sample containing SA into contact with the nucleic acid sensor of the present invention, and the catalytic nucleic acid molecule (D) in the nucleic acid sensor.
  • the sample is not particularly limited.
  • the sample may be, for example, either a sample containing SA or a sample whose SA is unknown.
  • the sample is preferably a liquid sample, for example.
  • the nucleic acid element When the nucleic acid element is used in a free state as the nucleic acid sensor of the present invention, it is preferable to bring the nucleic acid element and the sample into contact, for example, in a container such as a tube.
  • a container such as a tube.
  • the nucleic acid element of the present invention When the nucleic acid element of the present invention is used in a state where the nucleic acid element is disposed on the base material, for example, the sample can be brought into contact with the nucleic acid element on the base material.
  • the detection step for example, it is preferable to detect a signal generated by the catalytic function of the catalytic nucleic acid molecule (D).
  • the signal include an optical signal and an electrochemical signal.
  • the optical signal include a color development signal, a luminescence signal, and a fluorescence signal.
  • the signal is preferably generated from a substrate by the catalytic function of the catalytic nucleic acid molecule (D), for example. Therefore, the detection step is preferably performed, for example, in the presence of a substrate corresponding to the catalytic function of the catalytic nucleic acid molecule (D).
  • the substrate is, for example, a substrate that generates a colored, luminescent, or fluorescent product by the catalytic function, a colored, luminescent, or fluorescent substrate, and a generation in which the colored, luminescent, or fluorescent light is lost by the catalytic function.
  • the substrate that generates a product and a substrate that generates a product of different color, luminescence, or fluorescence depending on the catalytic function.
  • the catalytic function can be detected by visually confirming, for example, the presence or absence of color development, luminescence or fluorescence, or the change or intensity of color development, luminescence or fluorescence as a signal.
  • the catalytic function can be detected by measuring the absorbance, reflectance, fluorescence intensity, and the like as signals using an optical technique.
  • the catalytic function include the catalytic function of the oxidation-reduction reaction as described above.
  • the catalytic nucleic acid molecule (D) has a catalytic function for the oxidation-reduction reaction
  • a substrate capable of transferring electrons can be mentioned.
  • a product is generated from the substrate by the catalytic nucleic acid molecule (D), and electrons are transferred in the process.
  • This electron transfer can be detected electrochemically as an electrical signal by application to an electrode, for example.
  • the electric signal can be detected by measuring the intensity of the electric signal such as an electric current.
  • the substrate is not particularly limited, and for example, hydrogen peroxide, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 1,2-phenylenediamine (OPD), 2,2′-Azinobis (3-ethylbenzothiazole- 6-sulfonic Acid Ammonium Salt (ABTS), 3,3′-Diaminobenzodinine (DAB), 3,3′-Diaminobenzoidine Tetrahhydrochloridate Hydrate (DAB4HCl), 3-Amino-9-EC-9-EC (4C1N), 2,4,6-Tribromo-3-hydroxybenzoic Acid, 2,4-Dichlorophenol, 4-Aminoantipyrine, 4-Aminoantipyrine Hydrochloride, luminol and the like.
  • the substrate may be supplied to the nucleic acid sensor in advance, for example, before contacting the sample with the nucleic acid sensor, or at the same time as contacting the sample or after contacting the sample.
  • the nucleic acid sensor may be supplied.
  • the substrate is preferably supplied to the nucleic acid sensor, for example, as a substrate solution mixed with a liquid.
  • the liquid mixed with the substrate is preferably a buffer such as Tris-HCl.
  • the concentration of the substrate in the substrate solution is not particularly limited, and is, for example, 0.1 to 5 mmol / L, preferably 0.5 to 2 mmol / L.
  • the pH of the substrate solution is, for example, 6-9, preferably 6.8-9.
  • the reaction conditions with the catalytic nucleic acid molecule (D) are not particularly limited.
  • the temperature is, for example, 15 to 37 ° C.
  • the time is, for example, 10 to 900 seconds.
  • porphyrin may coexist in addition to the substrate.
  • Some known DNAzymes exhibit higher redox activity by forming a complex with porphyrin, for example. Therefore, in the present invention, for example, redox activity may be detected as a complex of the catalytic nucleic acid molecule (D) and porphyrin in the presence of porphyrin.
  • the supply of porferin is not particularly limited and can be performed in the same manner as the substrate.
  • the porphyrin is not particularly limited, and examples thereof include unsubstituted porphyrin and derivatives thereof.
  • examples of the derivatives include substituted porphyrins and metal porphyrins complexed with metal elements.
  • Examples of the substituted porphyrin include N-methylmesoporphyrin.
  • Examples of the metal porphyrin include hemin, which is a trivalent iron complex.
  • the porphyrin is, for example, preferably the metal porphyrin, more preferably hemin.
  • SA can be detected as described above.
  • biotin can be indirectly detected, and as a specific example, a biotinylated target or a binding concentration of biotin can be indirectly detected.
  • an SA aptamer that binds to the binding site of biotin is used as the binding nucleic acid molecule. Since the SA aptamer and biotin each bind to the same site of SA, the SA aptamer cannot bind when biotin binds to SA. For this reason, as the amount of biotin increases, the binding between the SA aptamer and SA is inhibited.
  • the amount of biotin or biotinylated target is determined by reacting the nucleic acid sensor of the present invention with SA and biotin or a biotinylated target and detecting the catalytic function of the catalytic nucleic acid molecule in the nucleic acid sensor of the present invention. It becomes possible to evaluate.
  • the analysis device of the present invention is an SA analysis device, and includes a base material, a nucleic acid sensor, and a detection unit.
  • the nucleic acid sensor and the detection unit are arranged on the base material, and the nucleic acid sensor is the book
  • the detection unit is a detection unit that detects a catalytic function of the catalytic nucleic acid molecule (D) in the nucleic acid sensor.
  • the analysis device of the present invention is characterized by using the nucleic acid sensor of the present invention, and the other configurations are not limited at all. Unless specifically described, the analysis device of the present invention can use, for example, the description of the nucleic acid sensor of the present invention.
  • the arrangement method of the nucleic acid sensor is not particularly limited.
  • the nucleic acid element in the nucleic acid sensor may or may not be immobilized on the base material. Also good.
  • the nucleic acid element may be directly fixed or indirectly fixed to the base material, for example.
  • the immobilization include linking by chemical bonding.
  • Examples of the indirect immobilization include a form in which the nucleic acid element is immobilized on the base material via a linker.
  • the linker include the terminal linkers described above.
  • nucleic acid immobilization method for example, a known nucleic acid immobilization method can be adopted.
  • the method include a method using photolithography, and specific examples thereof can be referred to US Pat. No. 5,424,186.
  • the nucleic acid immobilization method include a method of synthesizing the nucleic acid element on the base material. As this method, for example, a so-called spot method can be mentioned.
  • US Pat. No. 5,807,522 Japanese Patent Publication No. 10-503841 and the like can be referred to.
  • the arrangement site of the nucleic acid element in the substrate is not particularly limited, and examples thereof include a form arranged in the detection unit.
  • the analysis device of the present invention may further have a reagent part, for example.
  • the reagent unit may be disposed in the detection unit.
  • a reagent may be arranged in advance in the reagent unit, or the reagent may be supplied at the time of use.
  • the reagent include the aforementioned substrate and the porphyrin.
  • the detection unit is a detection unit that detects the catalytic function of the catalytic nucleic acid molecule (D).
  • the detection unit is preferably a detection unit that detects a signal generated by the catalytic function of the catalytic nucleic acid molecule (D) as the catalytic function of the catalytic nucleic acid molecule (D).
  • Examples of the signal include a signal from a substrate due to the catalytic function of the catalytic nucleic acid molecule (D), as described above.
  • Examples of the signal include an optical signal and an electrochemical signal as described above.
  • the detection unit is, for example, an optical signal detection unit, and examples include a detection unit such as absorbance, reflectance, and fluorescence intensity.
  • the detection unit has, for example, an electrode system.
  • the said detection part can be formed by arrange
  • the arrangement method of the electrodes is not particularly limited, and for example, a known method can be adopted. Specific examples include thin film forming methods such as vapor deposition, sputtering, screen printing, and plating.
  • the electrode may be disposed directly or indirectly on the substrate.
  • An indirect arrangement includes, for example, an arrangement through another member.
  • the electrode system may include, for example, a working electrode and a counter electrode, or may include a working electrode, a counter electrode, and a reference electrode.
  • the material of the electrode is not particularly limited, and examples thereof include platinum, silver, gold, and carbon.
  • Examples of the working electrode and the counter electrode include a platinum electrode, a silver electrode, a gold electrode, and a carbon electrode, and examples of the reference electrode include a silver / silver chloride electrode.
  • the silver / silver chloride electrode can be formed, for example, by laminating a silver chloride electrode on a silver electrode.
  • the nucleic acid element is preferably disposed, for example, in the electrode system, and is preferably disposed in the working electrode among the electrodes.
  • the analytical device of the present invention includes the electrode system and the reagent part, for example, the reagent part is preferably disposed on the electrode system.
  • the analysis device of the present invention may include a plurality of detection units, for example.
  • the analytical device fractionates the surface of the base material into a matrix, and includes a detection unit as described above in each fractionation region.
  • the number of nucleic acid sensors arranged in one detection unit is not particularly limited.
  • the substrate is not particularly limited.
  • the substrate is preferably a substrate having an insulating surface, for example.
  • the substrate may be, for example, a substrate made of an insulating material, or a substrate having an insulating layer made of an insulating material on the surface.
  • the insulating material is not particularly limited, and examples thereof include known materials such as glass, ceramic, insulating plastic, and paper.
  • the insulating plastic is not particularly limited, and examples thereof include silicone resin, polyimide resin, epoxy resin, and fluorine resin.
  • the analysis method of the present invention is an analysis method of SA, the contacting step of bringing a sample into contact with the analysis device of the present invention, and the catalytic nucleic acid molecule in the detection unit of the analysis device It includes a detection step of detecting SA in the sample by detecting the catalytic function of (D).
  • the analysis method of the present invention is characterized in that the analysis device including the nucleic acid sensor of the present invention is used, and other conditions are not limited at all.
  • the analysis method of the present invention for example, the description of the analysis method in the nucleic acid sensor of the present invention can be cited.
  • the analytical reagent of the present invention includes the nucleic acid sensor of the present invention.
  • the analytical reagent of the present invention is characterized by including the nucleic acid sensor, and other configurations are not limited at all.
  • the analytical reagent of the present invention may contain, for example, components such as the substrate, the poferrin, the buffer solution, and / or the substrate in addition to the nucleic acid sensor.
  • the analytical reagent of the present invention may be, for example, an analytical kit.
  • the nucleic acid sensor and the other components described above may be included and separately accommodated.
  • the analysis kit may further include instructions for use, for example.
  • Example 1 The single-stranded nucleic acid element (III) comprising SA aptamer as the binding nucleic acid molecule (A) and EAD2 as the catalytic nucleic acid molecule (D) was prepared, and the performance as a nucleic acid sensor was confirmed.
  • DNA having the following sequence was synthesized (see FIG. 4A).
  • the underlined portion on the 5 ′ side is EAD2 (D) of SEQ ID NO: 11
  • the underlined portion on the 3 ′ side is the SA aptamer (A) of SEQ ID NO: 5, with poly dT interposed therebetween. It was set as sequence (I).
  • SA.EAD2.D0.A0 SEQ ID NO: 59
  • the reaction was similarly performed for a reaction solution (PC) using EAD2 of SEQ ID NO: 11 instead of the nucleic acid sensor. Further, the reaction was performed in the same manner as described above except that the SA aptamer of SEQ ID NO: 5 was used as the negative control 1 instead of the nucleic acid sensor, and the nucleic acid sensor was excluded as the negative control 2 The same reaction was carried out for the reaction solution (W / O).
  • EAD2 SEQ ID NO: 11
  • CTGGGAGGGAGGGAGGGA SA aptamer SEQ ID NO: 5
  • FIG. 5 shows the results of color development of the reaction solution.
  • FIG. 5 (A) is a photograph showing the coloring of the reaction solution, and shows that the darker the color, the stronger the color development to blue.
  • FIG. 5B is a graph showing the absorbance of the reaction solutions having SA concentrations of 0 ⁇ mol / L and 10 ⁇ mol / L.
  • the color development of the reaction solution depends on the SA concentration because the color development becomes stronger as the SA concentration increases. I understood. For this reason, according to the nucleic acid sensor of an Example, it can be said that the presence and concentration of SA can be judged visually.
  • Example 2 The single-stranded nucleic acid element (II) comprising SA aptamer as the binding nucleic acid molecule (A) and DNAzyme as the catalytic nucleic acid molecule (D) was prepared, and the performance as a nucleic acid sensor was confirmed.
  • the nucleic acid element DNA having the following sequence was synthesized (see FIG. 2B).
  • the lower-case sequence is the stem-forming sequence (S A )
  • the upper-case poly dT is the loop-forming sequence (L2)
  • the underline is the DNAzyme (D) of SEQ ID NO: 18 (neco0584)
  • the lower case sequence was the stem forming sequence (S D )
  • the upper case poly dT was the loop forming sequence (L1)
  • the underlined portion was the SA aptamer (A) of SEQ ID NO: 5.
  • a reaction solution having the following composition 100 ⁇ L of a reaction solution having the following composition was prepared in an Eppendorf tube and reacted at 25 ° C. for 60 seconds, and then the absorbance of the reaction solution was measured (wavelength 415 nm). The measurement used the light absorbency measuring apparatus (Brand name TECAN infinite, TECAN company).
  • the composition of the DNAzyme buffer was 50 mmol / L Tris-HCl (pH 7.4), 20 mmol / L KCl, 0.05% Triton X-100.
  • ABTS (2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) was used as the substrate.
  • DNAzyme of SEQ ID NO: 18 was used as a positive control.
  • the reaction was similarly carried out for the reaction solution (W / O) excluding the nucleic acid sensor.
  • FIG. 6 is a graph showing the absorbance of the reaction solution.
  • the absorbance of the reaction solution containing 1 ⁇ mol / L of SA showed a significant difference from the absorbance of the reaction solution without addition of SA. From these results, it can be said that according to the nucleic acid sensor of the example, the presence or absence and concentration of SA can be measured by measuring absorbance, and specifically, it can be detected even with 1 ⁇ mol / L SA.
  • Example 3 The double-stranded nucleic acid element (I) having SA aptamer as the binding nucleic acid molecule (A) and DNAzyme as the catalytic nucleic acid molecule (D) was prepared, and the performance as a nucleic acid sensor was confirmed.
  • the 5 ′ underline is the SA aptamer (A) of SEQ ID NO: 5
  • poly dT is the loop-forming sequence (L1)
  • the 3 ′ underline is the DNAzyme of SEQ ID NO: 18 (neco0584).
  • SA.neco.D3A2 SEQ ID NO: 32
  • DNA having the following sequence was synthesized as the second strand (ss2) with respect to the first strand (ss1) (see FIG. 1A).
  • the underlined portion on the 5 ′ side is the stem-forming sequence (S D ) complementary to the 5′-side region of the DNAzyme of the first strand (ss1)
  • poly dT is the loop-forming sequence (L2
  • the underlined part on the 3 ′ side was the stem-forming sequence (S A ) complementary to the 3 ′ side region of the SA aptamer of the first strand (ss1).
  • SA.neco.D8.A5 (SEQ ID NO: 45) 5'- TCCCACCC TTTTTTTT CCGAA -3 '
  • a reaction solution having the following composition was prepared and reacted at 25 ° C. for 60 seconds, and then the absorbance of the reaction solution was measured (wavelength 415 nm). The measurement used the light absorbency measuring apparatus (Brand name TECAN infinite, TECAN company).
  • the composition of the DNAzyme buffer was 50 mmol / L Tris-HCl (pH 7.4), 20 mmol / L KCl, 150 mmol / L, 0.05% Triton X-100. ABTS was used as the substrate.
  • composition of reaction solution 1 ⁇ mol / L first strand (ss1) 2 ⁇ mol / L second strand (ss2) 3 ⁇ mol / L Hemin 1 ⁇ mol / L SA 50 mmol / L DNAzyme buffer 1 mmol / L substrate 0.5 mmol / L H 2 O 2
  • FIG. 7 is a graph showing the absorbance of the reaction solution.
  • the absorbance of the reaction solution containing 1 ⁇ mol / L SA showed a significant difference from the absorbance of the reaction solution without addition of SA. From these results, it can be said that according to the nucleic acid sensor of the example, the presence or absence and concentration of SA can be measured by measuring absorbance, and specifically, it can be detected even with 1 ⁇ mol / L SA.
  • ON / OFF of the catalytic function of the catalytic nucleic acid molecule (D) can be switched depending on whether or not the binding nucleic acid molecule (A) and SA are bound. Therefore, the presence or amount of SA can be easily detected by detecting the catalytic function of the catalytic nucleic acid molecule.
  • the analysis device of the present invention uses the nucleic acid sensor as described above, for example, the device can be reduced in size and chipped, and a simple analysis can be performed even for a large number of samples. . For this reason, the present invention can be said to be an extremely useful technique for research and examination in various fields such as clinical medicine, food, and environment.

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Abstract

L'invention concerne un nouveau capteur pour détecter la streptavidine (SA). Cet acide nucléique capteur pour l'analyse de SA comprend un élément d'acide nucléique ayant une molécule d'acide nucléique catalytique (D) présentant une fonction catalytique, et une molécule d'acide nucléique de liaison (A) qui se lie à SA. L'élément d'acide nucléique est un élément d'acide nucléique double brin configuré par un premier brin et un second brin. Le premier brin (ss1) a la molécule d'acide nucléique de liaison (A), une séquence de formation de boucle (L1), et la molécule d'acide nucléique catalytique (D) liées dans cet ordre. Le second brin (ss2) a une séquence de formation de tige (Sa), une séquence de formation de boucle (L2) et une séquence de formation de souches (Sd) liées, dans cet ordre. La fonction catalytique de la molécule d'acide nucléique catalytique (D) est inhibée dans l'élément d'acide nucléique par la formation de tige par à la fois les séquences de formation de tige (SA, SD), en l'absence de SA. La formation de tige est annulée en présence de l'Acide Salicylique (SA), en tant que résultat de la liaison entre la molécule d'acide nucléique de liaison (A) et SA et la fonction catalytique de la molécule d'acide nucléique catalytique (D) est présentée.
PCT/JP2013/058042 2012-03-23 2013-03-21 Dispositif pour l'analyse cible de la streptavidine, et procédé d'analyse WO2013141291A1 (fr)

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US14/387,431 US9880161B2 (en) 2012-03-23 2013-03-21 Device and method for analyzing streptavidin
EP13763690.8A EP2829604A4 (fr) 2012-03-23 2013-03-21 Dispositif pour l'analyse cible de la streptavidine, et procédé d'analyse
JP2014506266A JP6183917B2 (ja) 2012-03-23 2013-03-21 ストレプトアビジンの分析用デバイスおよび分析方法

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WO2017098746A1 (fr) * 2015-12-11 2017-06-15 Ｎｅｃソリューションイノベータ株式会社 Détecteur destiné à l'analyse du cortisol, procédé d'analyse du cortisol, réactif d'évaluation du stress, procédé d'évaluation du stress, réactif de test destiné à une maladie liée au cortisol, et procédé de test destiné au risque de contraction d'une maladie liée au cortisol
WO2017188426A1 (fr) * 2016-04-28 2017-11-02 国立大学法人東京農工大学 Aptamère améliorant l'activité de peroxydase
JP2017200472A (ja) * 2016-04-28 2017-11-09 国立大学法人東京農工大学 ペルオキシダーゼ活性増大アプタマー
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JP2020039300A (ja) * 2018-09-11 2020-03-19 国立研究開発法人物質・材料研究機構 酸化還元活性を有する核酸分子

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JPWO2015083391A1 (ja) * 2013-12-04 2017-03-16 Ｎｅｃソリューションイノベータ株式会社 ピーナッツに結合する核酸分子およびその用途
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WO2015151349A1 (fr) * 2014-03-31 2015-10-08 Ｎｅｃソリューションイノベータ株式会社 Molécule d'acide nucléique se liant à un allergène du sarrasin et utilisation s'y rapportant
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